In poultry production, antibiotics used as growth promoters (AGPs) improve feed intake, weight gain, and feed efficiency. A meta-analysis including 121,643 broilers found that AGP-supplemented diets increased weight gain and improved feed conversion ratio in the initial (1–21 d) and total (1–42 d) phases, resulting in an estimated economic benefit of US$ 0.03 per bird, equivalent to US$ 183.56 million annually (Cardinal et al., 2019). However, their overuse raises public health concerns due to the emergence of antibiotic-resistant bacteria (Iwu et al., 2020). Consequently, alternative nutritional strategies have been developed for antibiotic-free systems (Hernandez-Platan et al., 2023). Phytogenic additives have gained attention for their antimicrobial (Mnisi et al., 2023), antioxidant (Akosile et al., 2023), immunomodulatory (Albarrak et al., 2021), and gut-health-promoting effects (Yang et al., 2021).

The black wattle (Acacia mearnsii) extract (BWE) is rich in condensed tannins, consisting of flavan-3-ols or flavan-3,4-diols units (Godoy et al., 2024). These tannins are mainly proanthocyanidins composed of robinetinidol and fisetinidol extension units, with catechin and gallocatechin as terminal units, and contain minor amounts of phenolic acids such as gallic acid (Ogawa et al., 2018). Detailed chemical profiling has shown that approximately 77% of BWE consists of oligomeric proanthocyanidins, with varying degrees of polymerization and structural subtypes identified by high-performance liquid chromatography (HPLC) and mass spectrometry (Chen et al., 2018).

In vitro assays already have demonstrated that BWE exhibits antimicrobial activity against Campylobacter jejuni (Kurekci et al., 2012). However, this extract did not reduce C. jejuni counts in cecal contents or feces when assessed an in vivo assay in broilers (Kurekci et al., 2014). Recent findings indicate that up to 500 mg kg⁻¹of acacia tannins may mitigate the effects of Salmonella Heidelberg and necrotic enteritis on broiler performance, improving digestibility and intestinal integrity and reducing Salmonella in the cecal content of broilers (Godoy et al., 2024; Maysonnave et al., 2024). Other tannin-rich extracts have been investigated, yielding promising results for broilers, particularly in alleviating oxidative stress associated with Eimeria spp. infections. (Wang et al., 2008), and resulting in decreased oocyst counts (Perin et al., 2019).

However, the antinutritional effects of tannins in broiler diets are well-documented, often resulting in decreased performance due to reduced feed intake and impaired digestibility of minerals and proteins, caused by the formation of nutrient-binding complexes (Garcia et al., 2004; Hassan et al., 2020). A level of 900 mg kg⁻¹ leads to a reduction in performance and digestibility (Godoy et al., 2024), while the inclusion of 500 mg kg⁻¹ of tannins in poultry diets resulted in reduced bone mineralization (Bayerle et al., 2019).

The impact of tannins on health and performance depends on their chemical structure and dosage, with evidence suggesting that moderate levels may enhance nutrient utilization and overall bird health (Redondo et al., 2014; Godoy, 2024). Thus, this study aimed to evaluate increasing levels of black wattle bark extract (BWE) in broilers’ diets and their effects on performance, nutrient digestibility, intestinal health, and bone quality. We hypothesized that moderate levels of BWE would improve performance and health parameters without eliciting antinutritional effects.

The trial was conducted in accordance with the National Council for Animal Control and Experimentation (CONCEA) rules and approved by the Ethics Committee on Animal Use of the Instituto de Zootecnia of the Universidade Federal Rural do Rio de Janeiro, under protocol number 0118-05-2021.

From 8 to 14 days of age, dietary supplementation with BWE produced no significant changes in feed intake, body weight gain, or feed conversion ratio (P>0.05; Table 2). Between days 15 and 21 (Table 2), feed intake decreased linearly with increasing BWE inclusion. Tukey’s test confirmed a dose-dependent effect, with birds fed 300 mg kg⁻¹ showing the highest intake and those at 700 and 900 mg kg⁻¹ showing the lowest values. For broilers from 22 to 28 days, the reduction in feed intake was accompanied by a decrease in weight gain (P<0.05), with lower weight gain in the groups fed 900 mg kg⁻¹, while broilers receiving 0 and 300 mg kg⁻¹of acacia tannins showed greater weight gain.

Table 2

	BWE inclusion level (mg kg−1)					SEM	P-value	Regression
	0	300	500	700	900
Day 8 to 14
Feed intake (g)	453.7	443.5	443.5	428.9	428.2	8.59	0.563	NS
Weight gain (g)	326.2	324.6	307.6	316.0	310.0	5.19	0.604	NS
Feed:gain ratio	1.390	1.367	1.394	1.405	1.382	0.021	0.595	NS
Day 15 to 21
Feed intake (g)	643.3ab	669.2a	631.7ab	613.1b	614.9b	10.81	0.013	L1
Weight gain (g)	476.2	480.0	461.9	473.2	457.4	7.12	0.461	NS
Feed:gain ratio	1.351	1.394	1.370	1.327	1.346	0.017	0.070	NS
Day 22 to 28
Feed intake (g)	1007ab	1021a	975.1ab	985.6ab	959.8b	17.21	0.006	L2
Weight gain (g)	716.2a	711.3a	684.8ab	696.5ab	669.7b	15.89	0.009	L3
Feed:gain ratio	1.407	1.438	1.425	1.415	1.435	0.017	0.666	NS
Day 8 to 28
Feed intake (g)	2104ab	2134a	2035ab	2042ab	2003b	28.87	0.021	L4
Weight gain (g)	1519	1516	1454	1485	1437	23.33	0.072	NS
Feed:gain ratio	1.386	1.408	1.401	1.373	1.394	0.010	0.138	NS

SEM - standard error of the mean; L - linear effect; NS - non-significant.

Considering the entire growth cycle (8 to 28 days), only feed intake was significantly influenced by the treatments (P = 0.02), with a linear reduction as the inclusion level of BWE increased. Broilers fed 300 mg kg⁻¹ of BWE exhibited the highest cumulative feed intake, whereas those receiving 900 mg kg⁻¹ showed the lowest values throughout the evaluated period. It is important to note that performance was evaluated only up to 28 of age under cage housing conditions, which limits direct extrapolation to commercial grow-out conditions typically extending to 42 days.

The BWE inclusion in broilers’ diets resulted in a linear response in dry matter and protein digestibility (Table 3), and a quadratic response was observed for ash retention. The proposed quadratic model estimated 40.77% as the maximum ash retention at an inclusion level of 495 mg kg⁻¹ of BWE.

Table 3

	BWE inclusion level (mg kg−1)					SEM	P-value	Regression
	0	300	500	700	900
ATTDDM (%)	69.03b	71.94a	71.34ab	70.73ab	71.76ab	0.673	0.034	L1
ATTDCP (%)	63.28c	66.88ba	68.64a	64.44cb	68.57a	0.859	<0.01	L2
ARC (%)	27.53c	39.67a	44.23a	29.35cb	32.96b	1.310	<0.01	Q3
AME (kcal kg−1)	3227	3277	3326	3265	3312	32.33	0.242	NS
AMEn (kcal kg−1)	2995	3029	3050	3031	3049	30.30	0.706	NS

ATTDDM - apparent total tract digestibility of dry matter; ATTDCP - ATTD of crude protein; ARC - ash retention coefficient; AME - apparent metabolizable energy; AMEn - nitrogen-corrected apparent metabolizable energy; SEM - standard error of the mean; L - linear; Q - quadratic; NS - non-significant.

Despite the significant linear effect, a low coefficient of determination was observed. The Tukey’s HSD test (Table 3) showed a higher dry matter digestibility value when 300 mg kg⁻¹ of BWE was supplied compared with the treatment without BWE inclusion. For protein digestibility, the best value was observed with the inclusion of 500 and 900 mg kg⁻¹ of BWE. Generally, the highest digestibility values were observed at inclusion levels up to 500 mg kg⁻¹ concentrations. Dietary values of AME and AMEn were not affected (P>0.05) by the BWE inclusion in broiler diets.

No differences (P>0.05) were found in the total “I See Inside” index (Table 4) in the ileum of broilers slaughtered at 21 of age. In the analyzed indices of intestinal health, only inflammatory cell infiltration in the epithelium, goblet cells, and oocyst presence showed significant alterations (P<0.05). The broiler group fed 900 mg kg⁻¹ of BWE showed the worst results (P<0.05) for inflammatory cell infiltration in the epithelium compared with animals receiving only 300 mg kg⁻¹ of the additive. In addition, the animals fed with the diet with the lowest BWE concentration had higher goblet cell counts than those supplied with 500 mg kg⁻¹ of BWE. Finally, the group fed 900 mg kg⁻¹ of BWE showed a higher presence of oocysts than all others.

Table 4

Variable	BWE inclusion level (mg kg−1)					SEM	P-value
	0	300	500	700	900
Own lamina thickness	1.01	0.88	1.11	0.79	0.96	0.11	0.562
Epithelium thickness	0.53	0.46	0.60	0.64	0.60	0.06	0.174
Enterocyte proliferation	0.48	0.43	0.59	0.57	0.60	0.05	0.143
Inflammatory cell infiltration into epithelium	0.38ab	0.31b	0.47ab	0.45ab	0.56a	0.05	0.011
Inflammatory cell infiltration in the lamina propria	0.83	0.70	1.04	0.69	0.99	0.11	0.227
Goblet cells	0.72ab	0.50ab	0.47b	0.91a	0.91a	0.10	<0.01
Congestion	0.04	0.08	0.01	0.08	0.07	0.03	0.578
Presence of oocysts	0.00b	0.00b	0.02b	0.00b	0.17a	0.02	<0.01
Total score	3.99	3.36	4.34	4.13	4.86	0.40	0.247

SEM - standard error of the mean.

Keeping in view the bone parameters (Table 5), adding BWE in broiler feed did not influence (P>0.05) any of the bone quality characteristics in tibia.

Table 5

Variable	BWE inclusion level (mg kg−1)					SEM	P-value	Regression
	0	300	500	700	900
Seedor index	77.47	77.96	73.95	75.21	77.09	2.243	0.686	NS
Breaking strength (kgf)	21.32	18.27	19.53	18.00	20.68	1.531	0.466	NS
Ash (g)	1.11	1.10	1.10	1.12	1.14	0.046	0.967	NS
Dry matter content (%)	44.54	43.35	44.98	45.38	45.72	0.567	0.091	NS
Ash content (%, in dry matter)	47.93	48.74	49.65	50.17	50.33	0.695	0.102	NS

NS - non-significant (P>0.05) (n = 8); SEM - standard error of the mean.

The inclusion of black wattle extract (BWE) suppressed feed intake in a dose-dependent manner, with birds fed the highest level (900 mg kg⁻¹) consuming less than those fed 300 mg kg⁻¹. This finding is consistent with the well-known astringent properties of tannins, which can reduce palatability and feed acceptance (Butler et al., 1984). Meta-analyses by Soares et al. (2020) and Hidayat et al. (2021) have consistently associated tannin supplementation to reduced feed intake, weight gain, and feed conversion ratio.

A key finding of this study was the improvement in crude protein and dry matter digestibility, as well as ash retention at BWE inclusion levels up to 500 mg kg⁻¹. This result is particularly interesting given that tannins are known for their protein and metal binding capacity, which is typically associated with reduced amino acid digestibility (Hidayat et al., 2021), inhibition of digestive enzymes (Medugu et al., 2012) and reduced mineral retention by birds (Hassan et al., 2020). Godoy et al. (2024) reported similar improvements in protein digestibility in broilers receiving 500 mg kg⁻¹ of acacia tannins under a necrotic enteritis challenge. Antioxidant and anti-inflammatory effects of tannins may counteract their chelating properties, allowing reduced epithelial damage and better utilization of nutrients in the diet (Choi and Kim, 2020).

Despite improved dry matter and protein digestibility, these changes did not translate into better dietary energy utilization. This discrepancy may be explained by the fact that the improvements in digestibility were more pronounced in nitrogenous and mineral fractions rather than in primary energy-yielding components, such as fats or carbohydrates.

From a pathophysiological standpoint, the Eimeria challenge induces oxidative stress and intestinal inflammation (Masood et al., 2013). Tannins are potent antioxidants, with a reduction potential exceeding that of vitamins E and C (Shi et al., 2003). In this context, we observed a lower infiltration of inflammatory cells in the epithelium of birds supplemented with BWE300 compared with those receiving BWE900, indicating a beneficial, dose-dependent anti-inflammatory effect. This aligns with Tonda et al. (2018), who reported lower intestinal lesion scores in birds fed tannin extracts, and with Godoy et al. (2024), who reported improved villus height.

However, BWE supplementation did not alter the total intestinal lesion score (ISI) in our study. While the ISI methodology is highly correlated with performance (Belote et al., 2019), the lack of effect on both ISI and performance suggests that the tested BWE levels were safe but insufficient to promote significant gut integrity recovery or growth promotion under our specific challenge conditions. The overall ISI scores were relatively low compared with those reported by Belote et al. (2019), possibly because housing birds in metabolic cages limited oocyst reinfection, thereby diminishing the severity of the challenge compared with floor-pen systems.

Age may be another factor contributing to the different outcomes observed. In our study, we challenged birds at 8 days of age, whereas Belote et al. (2019) challenged birds that were only at 1 day of age. The developing immune system and gut barrier of neonatal chicks may respond more severely to an Eimeria challenge, potentially explaining the milder lesion scores recorded in our trial. Lima et al. (2025), for instance, reported that this same 10x challenge model, performed in metabolic cages, induces age-dependent changes in the expression of genes related to intestinal integrity. Therefore, analyzing these genes should be considered in future studies to confirm the gut health-promoting effects of tannins at a molecular level, especially when gross lesions are not pronounced.

Although an increase in ash retention was observed, this did not translate into improvements in tibia bone quality parameters. This apparent contradiction highlights the complex mechanisms involved in bone and mineral homeostasis. Bone deposition is not a passive outcome of mineral availability but is tightly controlled by parathyroid hormone (PTH), vitamin D, calcitonin, and fibroblast growth factor-23, as well as by the activity of osteoblasts and osteoclasts (Shao et al., 2019). Future studies should explore these variables to better understand the role of tannins in mineral metabolism after absorption.

While improved gut health can enhance mineral absorption, tannins are also known to chelate metals, which can reduce their bioavailability and impair bone mineralization (Hassan et al., 2020). Deleterious effects of tannins on bone quality have been previously reported (Keshavarzi et al., 2017; Ghaedi et al., 2018; Bayerle et al., 2019). In our study, the inclusion of acacia tannins up to 900 mg kg⁻¹ did not impair tibia mineralization or breaking strength. These findings reinforce that the impact of tannins on skeletal development is strongly dose- and source-dependent, with adverse effects occurring only when mineral binding is sufficient to alter mineral deposition.

The dietary inclusion of black wattle extract at levels up to 500 mg kg⁻¹ effectively improved nutrient digestibility and intestinal health in broilers under an Eimeria challenge, positioning it as a promising functional feed additive, particularly for antibiotic-free production systems. However, the intake suppression observed at 900 mg kg⁻¹ underscores that the optimal dose represents a critical balance between the beneficial effects and the inherent antinutritional properties of tannins.